Difference between revisions of "Team:Cornell/Composite Part"

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                                     <li><a href="https://2018.igem.org/Team:Cornell/Basic_Part">BASIC PARTS</a></li>
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                                    <li><a href="https://2018.igem.org/Team:Cornell/Composite_Part">COMPOSITE PARTS</a></li>
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         <!------------------------ COMPOSITE PAGE BANNER END ------------------------>
 
         <!------------------------ COMPOSITE PAGE BANNER END ------------------------>
  
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         <!------------------------ STANDARD PAGE SIDE BAR + CONTENT START ------------------------>
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                 <p class="composite-body-text">We are a team composed entirely of undergraduates and we pride ourselves on our independence. The concept of a biological band-pass filter based on frequency was brainstormed and implemented by our team. Our wet lab team designed and cloned all sequences into pSB1C3, and transformed them for testing in DH5-α E. Coli. They also designed all experiments and worked to analyze all the data we collected. The product development group worked to create the mathematical model for the system based on Hill Kinetics. The sub-team worked on both the deterministic and stochastic model. The policy and practices team spearheaded our outreach efforts and led initiatives to contact and work with synthetic biology researches on our campus and beyond. To understand how our project might integrate into the field, they conducted interviews to find out about how developing technologies enter a new field.</p>
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                <ul class="standard-page-side-bar">
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                     <li><a href="#reporterconstructs">REPORTER CONSTRUCTS</a></li>
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                    <li><a href="#combinationconstructs">COMBINATION CONSTRUCTS</a></li>
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                </ul>
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                    <p class="standard-page-content-text">
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                        This year, we are proud to present a variety of parts that ultimately allow for control of gene expression using specific frequency-based signals. The final gene products of these constructs can be replaced by any gene of interest, making these parts a versatile platform for precisely regulating a gene’s expression in multi-plasmid or construct systems. We have submitted BBa_K2561003 for consideration for best composite part.
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                    </p>
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                <div class="standard-page-content-section-last">
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                    <div class="standard-page-content-title"><div id="reporterconstructs">Reporter Constructs</div></div>
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                    <hr class="yellow-accent-line-left">
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                        These two composite parts were used to demonstrate the oscillatory nature of our genetic circuit. While incubated at 37 degrees Celsius, cells expressing the low pass reporter construct would express sfGFP (the reporter molecule), while cells expressing the high pass reporter construct would cease expression of sfGFP (normally constitutively expressed)
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                    </p>
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                    <div class="standard-page-content-subheading">BBa_K2561005: High Pass Reporter Construct</div>
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                    <br>
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                    <p class="standard-page-content-text">A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream reporter products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of a sfGFP reporter molecule that is normally constitutively expressed under the pTet promoter (BBa_R0040). TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations. </p>
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                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/f/fc/T--Cornell--demonstrateFigure1b.png" id="composite-img">
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                <div class="standard-page-content-section">
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                    <div class="standard-page-content-subheading">BBa_K2561003: Low Pass Reporter Construct</div>
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                    <br>
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                    <p class="standard-page-content-text">A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (Sigma F, BBa_K2561001) that in turn directs the expression of downstream reporter products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The Sigma F produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence (to direct expression of sfGFP, a reporter molecule. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations.</p>
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                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/b/bd/T--Cornell--demonstrateFigure1a.png" id="composite-img">
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                </div>
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                <div class="standard-page-content-section-last">
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                    <div class="standard-page-content-title"><div id="combinationconstructs">Combination Constructs</div></div>
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                    <hr class="yellow-accent-line-left">
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                    <div class="standard-page-content-subheading"></div>
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                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/5/57/T--Cornell--CombinationConstructSchematics.jpg" id="composite-img" style="height: 250px;">
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                    <p class="standard-page-content-text">
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                        The following two parts were designed to be be cotransformed into the same cell to control expression of a gene of interest under the control of the sigma-54-dependent <i>hrpL</i> promoter.  
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                    </p>
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                    <div class="standard-page-content-subheading">BBa_K2561006: High Pass Combination Construct (AND gate)</div>
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                    <p class="standard-page-content-text">
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                        A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream gene products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of HrpR, an enhancer-binding protein that forms a heteromeric complex with HrpS to regulate expression of the sigma-54-dependent <i>hrpL</i> promoter. TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations. This part should be used in conjunction with the Low Pass Combination Construct (AND gate) part (BBa_K2561004).
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                    </p>
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                    <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/5/51/T--Cornell--demonstrateFigure1d.png" id="composite-img" style="padding-bottom: 0">
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                    <div class="standard-page-content-subheading">BBa_K2561004: Low Pass Combination Construct (AND gate)</div>
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                    <p class="standard-page-content-text">
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                        A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (SigmaF) that in turn directs the expression of downstream gene products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The SigmaF produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence to direct expression of HrpS, an enhancer-binding protein that forms a heteromeric complex with HrpR to regulate expression of the sigma-54-dependent <i>hrpL</i> promoter. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations.This part should be used in conjunction with the High Pass Combination Construct (AND gate) part (BBa_K2561006).
 +
                    </p>
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                     <img class="standard-page-content-image" src="https://static.igem.org/mediawiki/2018/a/ab/T--Cornell--demonstrateFigure1c.png" id="composite-img" style="padding-bottom: 0">
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Latest revision as of 02:08, 18 October 2018

Team:Cornell/Composite - 2018.igem.org

Composite Part

This year, we are proud to present a variety of parts that ultimately allow for control of gene expression using specific frequency-based signals. The final gene products of these constructs can be replaced by any gene of interest, making these parts a versatile platform for precisely regulating a gene’s expression in multi-plasmid or construct systems. We have submitted BBa_K2561003 for consideration for best composite part.

Reporter Constructs

These two composite parts were used to demonstrate the oscillatory nature of our genetic circuit. While incubated at 37 degrees Celsius, cells expressing the low pass reporter construct would express sfGFP (the reporter molecule), while cells expressing the high pass reporter construct would cease expression of sfGFP (normally constitutively expressed)

BBa_K2561005: High Pass Reporter Construct

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream reporter products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of a sfGFP reporter molecule that is normally constitutively expressed under the pTet promoter (BBa_R0040). TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations.

BBa_K2561003: Low Pass Reporter Construct

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (Sigma F, BBa_K2561001) that in turn directs the expression of downstream reporter products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The Sigma F produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence (to direct expression of sfGFP, a reporter molecule. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this allows fluorescence signal to return to basal levels between oscillations.

Combination Constructs

The following two parts were designed to be be cotransformed into the same cell to control expression of a gene of interest under the control of the sigma-54-dependent hrpL promoter.

BBa_K2561006: High Pass Combination Construct (AND gate)

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of TetR (BBa_C0040) that in turn directs the expression of downstream gene products. Translation of TetR is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The TetR produced represses the expression of HrpR, an enhancer-binding protein that forms a heteromeric complex with HrpS to regulate expression of the sigma-54-dependent hrpL promoter. TetR is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations. This part should be used in conjunction with the Low Pass Combination Construct (AND gate) part (BBa_K2561004).

BBa_K2561004: Low Pass Combination Construct (AND gate)

A composite part consisting of a constitutively-active promoter sequence (J23100) that drives the expression of an orthogonal sigma factor (SigmaF) that in turn directs the expression of downstream gene products. Translation of Sigma F is additionally controlled by an upstream RNA thermometer construct that prevents recognition and binding of the RBS at temperatures lower than 32 degrees Celsius. The SigmaF produced can associate with the core RNA polymerase and bind the downstream PF2 promoter sequence to direct expression of HrpS, an enhancer-binding protein that forms a heteromeric complex with HrpR to regulate expression of the sigma-54-dependent hrpL promoter. Sigma F is tagged with a degradation tag (BBa_K2333405) characterized by the 2017 William and Mary team that targets it for degradation by the mf-Lon protease; this prevents leaky expression signals between oscillations.This part should be used in conjunction with the High Pass Combination Construct (AND gate) part (BBa_K2561006).